When generating a digital copy of a slide, i.e. when it is digitized, the image of the slide is generated from image field to image field through a step by step movement of the slide or the camera relative to each other. As a result of the operation, a series of frames is obtained in digital format, wherein each frame corresponds to a respective image field. To create a complete digital image of the slide, the thus obtained frames are stitched together. As the extension of the image fields is generally a few μm in each spatial direction, it takes a significant amount of time to generate a digital copy of the whole slide.
Confocal imaging provides a peculiar modality of optical microscopy that is capable of separating focused and non-focused beams in the light path of the microscope by physical and/or optical techniques. This enables the possibility of studying such sections/samples, the thicknesses of which significantly exceed the depth of focus or operation range of the objective.
Several principles are known for the technical implementation, however, it is an inherent feature of all forms of implementation that they lead to a decrease in light intensity. When fluorescent markers widely applied in routine diagnostics are used, this forms a serious limiting factor, because the emission of such fluorescent markers is only a fraction of the intensity of the exciting light. Thus, to keep the amount of time required for the complete digitization of a slide within acceptable limits, special technical solutions should be applied.
U.S. Pat. Nos. 6,144,489 and 6,687,052 disclose a variant of said technical implementation: confocal microscopy based on the principle of so-called structured illumination. The core feature of this technique is that the sample arranged on the slide under study is illuminated through a light modulation mask, and then the light reflected by the sample is simultaneously imaged as both passing through the mask and as reflected from the same light modulation mask. A sharp confocal image free of blurred background is obtained by subtracting the image of reflected light from the image of transmitted light.
An advantage of confocal microscopes based on the principle of said structured illumination is that they have the highest achievable light-utilization/confocality index among techniques known nowadays, which makes them particularly suitable for examination of fluorescently marked samples.
As confocal imaging based on structured illumination is, according to the aforementioned, based on the comparison of an image reflected from the mask and one transmitted through it, a confocal unit operating on this principle records optical sample sections tilted relative to the examined slide due to the distorting effect of the mask on the light path. This does not pose a problem in conventional microscopy, because subsequent tilted images still create a spatial (3D) image, fitting/stitching together multiple frames recorded next to each other does not pose significant difficulty. However when digitization is carried out to generate a complete image of the slide, the edges of the images next to each other do not match in Z direction, thus the recorded images cannot be continuously fitted to each other.